L-type CaV1.2 voltage-gated calcium channels are essential for mediating muscle contraction, hormone release from endocrine cells, and activity-dependent gene expression in neurons. Activity-dependent gene expression is an important mechanism for coupling repeated synaptic input to lasting changes in the neurons. This neuronal-specific function of CaV1.2 channels might arise from brain-specific alternative splicing. The N- terminus of CaV1.2 is encoded by alternative first exons; exon 1a (e1a) is the cardiac isoform and exon1b (e1b) has a broad expression pattern and is the only isoform identified to date in neuronal tissue. Preliminary data suggest that in the presence of the splicin factor Nova-2, e1a is repressed in brain and therefore promotes e1b expression. I hypothesize that e1b is essential for facilitating intracellular calcium signaling through the MAPK pathway to induce activity-dependent gene expression. Preliminary RT-PCR experiments demonstrate aberrant inclusion of e1a-CaV1.2 in Nova-2 -/- brain tissue but not in wild-type (WT). I hypothesize that Nova-2 represses the cardiac isoform (e1a-CaV1.2) in brain. Aim 1 will characterize e1a and e1b expression in specific WT and Nova-2 -/- tissues in the nervous system (cerebellum, cortex, and hippocampus) and cardiac tissue through RT-PCR. Ribonuclease protection assays will be used to quantify expression levels. I hypothesize that the switch in splicing in Nova-2 -/- neurons will lead to a decrease in e1b containing transcripts and an increase in e1a transcripts. Nova-2 cDNA will be transfected into Nova-2 -/- neurons to examine if Nova-2 represses e1a expression. These experiments will determine if Nova-2 is both necessary and sufficient to repress e1a in brain. Aim 2 will address how e1a and e1b differentially affect the neuronal function of CaV1.2, to induce activity dependent gene expression. First, I will analyze the electrophysiological properties (such as current densities and activation/inactivation kinetics) of the e1a/e1b CaV1.2 isoforms transfected into tsA201 cells. I do not expect there to be a difference in channel properties, because I suspect the difference between the isoforms instead lies in the induction of the calcium signaling pathway necessary for activity-dependent gene expression. To address this, I will use a KCl depolarization assay to examine isoform efficiency at coupling membrane depolarization to CREB phosphorylation, a hallmark of activation of the MAPK pathway by L-type channels. I will use siRNA and injection of isoform specific cDNA to manipulate levels of e1a and e1b isoforms in WT neurons to specifically link differences observed in CREB phosphorylation to differences in e1a/e1b expression and not another alternatively spliced exon in CaV1.2 or other transcripts regulated by Nova-2. I hypothesize that the e1b isoform will more efficiently induce CREB phosphorylation. These experiments will advance our understanding of how neuronal specific isoforms of CaV1.2 contribute to essential neuronal functions.